Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr17:24059509-24059603 95bp CGGTATCCTTCCTCCGTGT TTCTCAGCACTCAGCATAGGAC
Mutant= 95 bp
Wild Type = 92 bp
TGCAGTGTCTGTGGCTGAGACACTTGCTGGAGAGTATGGCTTCCACACTGTGCTGGCATCTTCCAGTTCTCAACAGAAAGAGCCAGTAGCACAAACAGGCACCTTCCAAGCTGCAGAAAAAAATGTGTCAGTTATCTCCCAGTATCTGTGGGCTGAGACCCGGTATCCTTCCTCCGTGTGTCACCTGTAGtgaccgtccgtaagtatgtctagtgaactgcctggcattttcagtcctatgctgagtgctgagaactgaaaggtctgtgaggatgttaggttaagcctgacacaagaacctcaggttagaggcagggtgtggggactaggggcgtacctgctttggtctgatctagaagaagctcaggttagagacagggtgtggggatcagggacgtatctgctttggtctgatctagaagaagtcactttttttaacctctatttcaggagtgttggaagaagcagaattttacaatatcacctcactaataaaacttgtaaaggacaaaattagagaacgagacagcaaaatatcccaggtgagatgggtgactaatctgcctgtagcttctgggcttgctgtataagcaggtgtggcttctctgtgtgtgaccttggaccagaactgtcctatccccatcctggagccccaggccttgctcccttcctcatctgccatgtctgccatgaagggccatcagtggagataggcagttctgtggaagttgtgtgatgagtgcacagaggagaaggaggcctgctggggagagTGAAGATGCTGGAGAGCAGAGGCTCTGATGGGAGAATATCAAGACCTGATCTGCCAGTGCAAAGGCCCATGGCCAGCACAGCAGAGAGGAGAGCATCGAGGAGCTGGGAAGTAGGTGGCACAGATACTGGGCATCTGTGCAGTCACTGGGAGGAGGTGGGCTTCCCGGGACCTCTGCTGCCCAGCATGTGTGTGTGTTGTGAGTGC
A 583 bp deletion beginning at Chromosome 17 position 24,058,991 bp and ending after 24,059,573 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45965 | CGG TAT CCT TCC TCC GTG T | Common | A | |||
45966 | TTC TCA GCA CTC AGC ATA GGA C | Wild type Reverse | A | |||
45967 | TTG CAC TGG CAG ATC AGG T | Mutant Reverse | A | |||
45968 | Fluorophore-1 | ACC GTC CGT AAG TAT GTC TAG TGA AC | Quencher-1 | WT Probe | ||
45969 | Fluorophore-2 | CCT GTA GTG AAG ATG CTG GAG AG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
45965 | 0.40 uM |
45966 | 0.40 uM |
45967 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |